Nanovolume Kinase Inhibition Assay Using a Sol−Gel-Derived Multicomponent Microarray

Rupcich, Nicholas; Green, James R. A.; Brennan, John D.

doi:10.1021/ac051405a

Cited by 27 publications

(38 citation statements)

References 49 publications

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“…microarrays, 26 using nanoliter-volume assays 14,27 and highly parallel screening. 28 An emerging functional protein microarray platform involves the pin-printing of protein-doped silica sols onto functionalized surfaces.…”

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confidence: 99%

A Sol−Gel-Derived Acetylcholinesterase Microarray for Nanovolume Small-Molecule Screening

et al. 2010

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A fluorimetric acetylcholinesterase (AChE) assay was developed and characterized both in solution and with the enzyme entrapped in sol-gel-derived silica. The assay is based on a disulfide-thiol interchange reaction between the intramolecularly quenched dimeric dye BODIPY FL l-cystine and thiocholine generated by the AChE-catalyzed hydrolysis of acetylthiocholine (ATCh), which results in a brightly fluorescent monomeric product owing to the cleavage of the disulfide-coupled form of the dye. The new assay was validated by comparison with the Ellman assay performed under parallel conditions and was used in both kinetic and end point assays. The assay was extended to the fabrication of functional AChE microarrays using contact pin-printing of sol-gel-derived silica. A total of 392 sol-gel formulations were screened for gelation times and 192 of these were further evaluated for array fabrication on four different surfaces using a factor analysis approach. Of these, 66 sol-gel/surface combinations produced robust microarrays, while 26 sol-gel/surface combinations were identified that could produce highly active AChE microarrays. The Z' factor for the on-array assay using an optimal sol-gel/surface combination, which considers both signal variability and difference in signals between positive and negative controls, was determined to be 0.60, which is above the minimum level required for applicability to screening. By overprinting nanoliter volumes of solutions containing the dye, ATCh, and potential inhibitors, these microarrays could be used to screen two libraries of small molecules, one composed of newly synthesized alkaloids and another consisting of ∼1000 known bioactive compounds, both as discrete compounds and mixtures thereof, for activity against AChE. IC(50) values were obtained on microarrays for compounds showing significant inhibitory activity, demonstrating the utility of arrays for quantitative inhibition assays.

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A Sol−Gel-Derived Acetylcholinesterase Microarray for Nanovolume Small-Molecule Screening

et al. 2010

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“…Martin et al described the Pro-Q diamond phospho-sensor dye which recognises phosphopeptides with remarkably little cross reactivity and a low false-negative rate [142]. It was demonstrated that this dye could be applied to kinase profiling [97,143,144] and phosphatase profiling [74,75] using high density peptide microarrays. A very similar approach was reported using biotinylated zinc(II) chelate phosphate sensor N-(5-(2-(?…”

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confidence: 99%

Deciphering Enzyme Function Using Peptide Arrays

2011

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Enzymes are key molecules in signal-transduction pathways. However, only a small fraction of more than 500 human kinases, 300 human proteases and 200 human phosphatases is characterised so far. Peptide microarray based technologies for extremely efficient profiling of enzyme substrate specificity emerged in the last years. This technology reduces set-up time for HTS assays and allows the identification of downstream targets. Moreover, peptide microarrays enable optimisation of enzyme substrates. Focus of this review is on assay principles for measuring activities of kinases, phosphatases or proteases and on substrate identification/optimisation for kinases. Additionally, several examples for reliable identification of substrates for lysine methyl-transferases, histone deacetylases and SUMO-transferases are given. Finally, use of high-density peptide microarrays for the simultaneous profiling of kinase activities in complex biological samples like cell lysates or lysates of complete organisms is described. All published examples of peptide arrays used for enzyme profiling are summarised comprehensively.

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“…Nevertheless, a number of recent promising studies have used Pro-Q DPS for the detection of phosphorylation events on microarrays (Rupcich et al 2005;Pal et al 2006). The first attempts to detect phosphorylation status and kinase activity on microarrays using fluorescence detection were made by using ProQ-Diamond phosphoprotein strain (Pro-Q DPS).…”

Section: Establishment Of Protein Microarrays For Phosphorylation Stumentioning

confidence: 99%

Plant Proteomics

Dumas‐Gaudot¹,

Recorbet²

2007

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EditorsJozef Šamaj received his Ph.D. degree in Plant Physiology from the Comenius University in Bratislava, Slovakia. He completed three post-doctoral programmes supported by Eurosilva, the Alexander von Humboldt Foundation, and the EU Marie Curie Programme in the highly regarded laboratories of Alain Boudet in Toulouse, Dieter Volkmann in Bonn, and Heribert Hirt in Vienna. He worked on the cell biology of somatic embryogenesis, lignifi cation in tree species, arabinogalactan proteins, the cytoskeleton, and signalling proteins. Jozef Šamaj has co-edited three books and co-authored more than 75 research papers, reviews, and book chapters. He is a senior lecturer and group leader at the Institute of Cellular and Molecular Botany in Bonn, Germany, and senior researcher at the Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Nitra, Slovakia. His current research is focussed on the role of signalling components and the cytoskeleton in relation to the vesicular traffi cking during plant development and stress responses using integrated cell-biological and functional proteomics approaches. Jozef Šamaj Jay Thelen v Jay Thelen received his B.Sc. degree in Biology and Biochemistry from the University of Nebraska-Lincoln in 1993. He earned his Ph.D. from the University of Missouri-Columbia (UMC) studying the structure and regulation of plant mitochondrial pyruvate dehydrogenase complexes under the guidance of Douglas Randall. In 1999 he started a 3-year postdoctoral position in John Ohlrogge's lab at Michigan State University investigating the plastid acetyl-CoA carboxylase protein complex. He returned to UMC in 2002 as the Associate Director of a campus Proteomics Center. In 2004, he was promoted to Assistant Professor in the Biochemistry Department, a position he currently holds. He has authored or co-authored 35 research and review articles since 1994. His research interests are centered around the regulation of plant metabolism, particularly carbon assimilation in oilseeds, and multienzyme metabolic complexes. He is currently studying seed fi lling in numerous crop oilseeds, using various quantitative proteomics approaches. He is also investigating global phosphoprotein networks involved in seed development and is developing improved strategies for quantitative proteomics.

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Nanovolume Kinase Inhibition Assay Using a Sol−Gel-Derived Multicomponent Microarray

Cited by 27 publications

References 49 publications

A Sol−Gel-Derived Acetylcholinesterase Microarray for Nanovolume Small-Molecule Screening

A Sol−Gel-Derived Acetylcholinesterase Microarray for Nanovolume Small-Molecule Screening

Deciphering Enzyme Function Using Peptide Arrays

Plant Proteomics

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